1. Field of the Invention
The present invention concerns DNA isolated from a porcine reproductive and respiratory virus (PRRSV), a protein and/or a polypeptide encoded by the DNA, a vaccine which protects pigs from a PRRSV based on the protein or DNA, a method of protecting a pig from a PRRSV using the vaccine, a method of producing the vaccine, a method of treating a pig infected by or exposed to a PRRSV, and a method of detecting a PRRSV.
2. Discussion of the Background
In recent years, North American and European swine herds have been susceptible to infection by new strains of reproductive and respiratory viruses (see A.A.S.P., September/October 1991, pp. 7-11; The Veterinary Record, Feb. 1, 1992, pp. 87-89; Ibid., Nov. 30, 1991, pp. 495-496; Ibid., Oct. 26, 1991, p. 370; Ibid., Oct. 19, 1991, pp. 367-368; Ibid., Aug. 3, 1991, pp. 102-103; Ibid., Jul. 6, 1991; Ibid., Jun. 22, 1991, p. 578; Ibid., Jun. 15, 1991, p. 574; Ibid., Jun. 8, 1991, p. 536; Ibid., Jun. 1, 1991, p. 511; Ibid., Mar. 2, 1991, p. 213). Among the first of the new strains to be identified was a virus associated with the so-called Mystery Swine Disease (MSD) or “blue-eared syndrome”, now known as Swine Infertility and Respiratory Syndrome (SIRS) or Porcine Reproductive and Respiratory Syndrome (PRRS).
An MSD consisting of reproductive failure in females and respiratory disease in nursing and weaned pigs appeared in the midwestern United States in 1987 (Hill et al., Am. Assoc. Swine Practitioner Newsletter 4:47 (1992); Hill et al., Proceedings Mystery Swine Disease Committee Meeting, Denver, Colo. 29-31 (1990); Keffaber, Am. Assoc. Swine Practitioner Newsletter 1:1-9 (1989); Loula, Agri-Practice 12:23-34 (1991)). Reproductive failure was characterized by abortions, stillborn and weak-born pigs. The respiratory disease in nursing and weaned pigs was characterized by fever, labored breathing and pneumonia. A similar disease appeared in Europe in 1990 (Paton et al., Vet. Rec. 128:617 (1991); Wensvoort et al., Veterinary Quarterly 13:121-130 (1991); Blaha, Proc. Am. Assoc. Swine Practitioners, pp. 313-315 (1993)), and has now been recognized worldwide.
This disease has also been called porcine epidemic abortion and respiratory syndrome (PEARS), blue abortion disease, blue ear disease (U.K.), abortus blau (Netherlands), seuchenhafter spatabort der schweine (Germany), Heko-Heko disease, and in the U.S., Wabash syndrome, mystery pig disease (MPD), and swine plague (see the references cited above and Meredith, Review of Porcine Reproductive and Respiratory Disease Syndrome, Pig Disease Information Centre, Department of Veterinary Medicine, Madingley Road, Cambridge CB3 OES, U.K. (1992); Wensvoort et al., Vet. Res. 24:117-124 (1993); Paul et al., J. Clin. Vet. Med. 11:19-28 (1993)). In Europe, the corresponding virus has been termed “Lelystad virus.”
At an international conference in May, 1992, researchers from around the world agreed to call this disease Porcine Reproductive and Respiratory Syndrome (PRRS). The disease originally appeared to be mainly a reproductive disease during its early phases, but has now evolved primarily into a respiratory disease.
Porcine reproductive and respiratory syndrome virus (PRRSV) is a relatively recently recognized swine pathogen associated with porcine reproductive and respiratory syndrome (PRRS). PRRSV is a significant pathogen in the swine industry. PRRSV infections are common in the U.S. swine herds. Outbreaks of PRRS in England have led to cancellation of pig shows.
The symptoms of PRRS include a reluctance to eat (anorexia), a mild fever (pyrexia), cyanosis of the extremities (notably bluish ears), stillbirths, abortion, high mortality in affected litters, weak-born piglets and premature farrowing. The majority of piglets born alive to affected sows die within 48 hours. PRRS clinical signs include mild influenza-like signs, rapid respiration (“thumping”), and a diffuse interstitial pneumonitis. PRRS virus has an incubation period of about 1-2 weeks from contact with a PRRSV-infected animal. The virus appears to be an enveloped RNA arterivirus (The Veterinary Record, Feb. 1, 1992). The virus has been grown successfully in pig alveolar macrophages and CL2621 cells (Benfield et al, J. Vet. Diagn. Invest., 4:127-133, 1992; Collins et al, Swine Infertility and Respiratory Syndrome/Mystery Swine Disease. Proc., Minnesota Swine Conference for Veterinarians, pp. 200-205, 1991), and in MARC-145 cells (Joo, PRRS: Diagnosis, Proc., Allen D. Leman Swine Conference, Veterinary Continuing Education and Extension, University of Minnesota (1993), 20:53-55; Kim et al, Arch. Virol., 133:477-483 (1993)). A successful culturing of a virus which causes SIRS has also been reported by Wensvoort et al (Mystery Swine Disease in the Netherlands: The Isolation of Lelystad Virus. Vet. Quart. 13:121-130, 1991).
Initially, a number of agents were incriminated in the etiology of this disease (Wensvoort et al., Vet. Res. 24:117-124 (1993); Woolen et al., J. Am. Vet. Med. Assoc. 197:600-601 (1990)). There is now a consensus that the causative agent of PRRS is an enveloped RNA virus referred to as Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), reportedly of approximately 62 nm in diameter (Benfield et al., J. Vet. Diagn. Invest., 4:127-133, 1992).
Virus isolates vary in their ability to replicate in continuous cell lines. Some grow readily, while others require several passages and some grow only in swine alveolar (SAM) cultures (Bautista et al., J. Vet. Diagn. Invest. 5:163-165, 1993; see also the Examples hereunder [particularly Table 1]).
PRRSV is a member of an Arterivirus group which includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) and simian hemorrhagic fever virus (SHFV) (Benfield et al., 1992, supra; Plagemann, Proc. Am. Assoc. Swine Practitioners, 4:8-15 1992; Plagemann and Moennig, Adv. Virus Res. 41:99-192, 1992; Conzelmann et al., Virology, 193:329-339, 1993; Godney et al., Virology, 194:585-596, 1993; Meulenberg et al., Virology, 192:62-72, 1993). The positive-strand RNA viruses of this Arterivirus group resemble togaviruses morphologically, but are distantly related to coronaviruses and toroviruses on the basis of genome organization and gene expression (Plagemann et al., supra; Spaan et al., J. Gen. Virol. 69, 2939-2952 (1988); Strauss et al., Annu. Rev. Biochem. 42, 657-683 (1988); Lai, Annu. Rev. Microbiol. 44, 303-333 (1990); Snijder et al., Nucleic Acid Res. 18, 4535-4542 (1990)). The members of this group infect macrophages and contain a nested set of 5 to 7 subgenomic mRNAs in infected cells (Plagemann et al., supra; Meulenberg et al., Virology, 192, 62-72 (1993); Conzelmann et al., Virology, 193, 329-339 (1993); 15, 16, 17, 18, 19).
The viral genome of European isolates has been shown to be a plus stranded RNA of about 15.1 kb (Conzelmann et al., supra; Meulenberg et al., supra), and appears to be similar in genomic organization to LDV and EAV (Meulenberg et al., supra). However, no serological cross-reaction has been found among PRRSV, LDV and EAV (Goyal et al., J. Vet. Diagn. Invest., 5, 656-664 (1993)).
PRRSV was initially cultivated in swine alveolar macrophage (SAM) cell cultures (Pol et al., Veterinary Quarterly, 13:137-143, 1991; Wensvoort et al., Veterinary Quarterly, 13:121-130, 1991) and then in continuous cell lines CL2621 (Benfield et al., supra), MA-104, and MARC-145 (Joo, Proc. Allen D. Leman Swine Conference, pp. 53-55, 1993). The reproductive and respiratory disease has been reproduced with cell free lung filtrates (Christianson et al., Am. J. Vet. Res., 53:485-488, 1992; Collins et al., J. Vet. Diagn. Invest., 4:117-126, 1992; Halbur et al.,Proc. Central Veterinary Conference, pp. 50-59, 1993), and with cell culture-propagated PRRSV (Collins et al., supra, and Proc. Allen D. Leman Swine Conference, pp. 47-48, 1993).
Eight open reading frames (also referred to herein as “ORFs” or “genes”) have been identified in a European PRRSV isolate. The genes of this European isolate are organized similarly to that in coronavirus (Meulenberg et al., supra). A 3′-end nested set of messenger RNA has been found in PRRSV-infected cells similar to that in coronaviruses (Conzelmann et al., supra; Meulenberg et al., supra).
The ORF 1a and 1b at the 5′-half of the European PRRSV genome are predicted to encode viral RNA polymerase. The ORF's 2-6 at the 3′-half of the genome likely encode for viral membrane-associated (envelope) proteins (Meulenberg et al., supra). ORF6 is predicted to encode the membrane protein (M) based on its similar characteristics with the ORF 6 of EAV, ORF 2 of LDV, and the M protein of mouse hepatitis virus and infectious bronchitis virus (Meulenberg et al., Virology 192, 62-72 (1993); Conzelmann et al., Virology 193, 329-339 (1993); Murtaugh, Proc. Allen D. Leman Swine Conference, Minneapolis, Minn., pp. 43-45 (1993); Mardassi et al., Abstracts of Conference of Research Workers in Animal Diseases, Chicago, Ill., pp. 43 (1993)). The product of ORF 7 is extremely basic and hydrophilic, and is predicted to be the viral nucleocapsid protein (N) (Meulenberg et al., supra; Conzelmann et al., supra; Murtaugh, supra; Mardassi et al., supra and J. Gen. Virol., 75:681-685 (1994)).
Although conserved epitopes have been identified between U.S. and European PRRSV isolates using monoclonal antibodies (Nelson et al., J. Clin. Microbiol., 31:3184-3189, 1993), there is extensive antigenic and genetic variation both among U.S. and European isolates of PRRSV (Wensvoort et al., J. Vet. Diagn. Invest., 4:134-138, 1992). European isolates are genetically closely related, as the nucleotide sequence at the 3′-half of the genome from two European PRRSV isolates is almost identical (Conzelmann et al., supra; Meulenberg et al., supra).
Although the syndrome caused by PRRSV appears to be similar in the U.S. and Europe, several recent studies have described phenotypic, antigenic, genetic and pathogenic variations among PRRSV isolates in the U.S. and in Europe (Murtaugh, supra; Bautista et al., J. Vet. Diagn. Invest., 5, 163-165 (1993); Bautista et al., J. Vet. Diagn. Invest., 5, 612-614 (1993); Wensvoort et al., J. Vet. Diagn. Invest., 4, 134-138 (1992); Stevenson et al., J. Vet. Diagn. Invest., 5, 432-434 (1993)). For example, the European isolates grow preferentially in SAM cultures and replicate to a very low titer in other culture systems (Wensvoort, Vet. Res., 24, 117-124 (1993); Wensvoort et al., J. Vet. Quart., 13, 121-130 (1991); Wensvoort et al., J. Vet. Diagn. Invest., 4, 134-138 (1992)). On the other hand, some of the U.S. isolates have been shown to replicate well in SAM as well as in the continuous cell line CL2621 (Benfield et al., J. Vet. Diagn. Invest., 4, 127-133 (1992); Collins et al., J. Vet. Diagn. Invest., 4, 117-126 (1992)). Thus, phenotypic differences among U.S. isolates are observed, as not all PRRSV isolates isolated on SAM can replicate on the CL2621 cell line (Bautista et al., J. Vet. Diagn. Invest., 5, 163-165 (1993)).
A high degree of regional antigenic variation among PRRSV isolates may exist. Four European isolates were found to be closely related antigenically, but these European isolates differed antigenically from U.S. isolates. Further, three U.S. isolates were shown to differ antigenically from each other (Wensvoort et al., J. Vet. Diagn. Invest., 4, 134-138 (1992)). Animals seropositive for European isolates were found to be negative for U.S. isolate VR 2332 (Bautista et al., J. Vet. Diagn. Invest., 5, 612-614 (1993)).
U.S. PRRSV isolates differ genetically at least in part from European isolates (Conzelmann et al., supra; Meulenberg et al., supra; Murtaugh et al., Proc. Allen D. Leman Conference, pp. 43-45, 1993). The genetic differences between U.S. and European isolates are striking, especially since they are considered to be the same virus (Murtaugh, supra). Similar observations were also reported when comparing the Canadian isolate IAF-exp91 and another U.S. isolate VR 2332 with LV (Murtaugh, supra; Mardassi, supra). However, the 3′ terminal 5 kb nucleotide sequences of two European isolates are almost identical (Conzelmann et al., supra; Meulenberg et al., supra).
The existence of apathogenic or low-pathogenic strains among isolates has also been suggested (Stevenson, supra). Thus, these studies suggest that the PRRSV isolates in North America and in Europe are antigenically and genetically heterogeneous, and that different genotypes or serotypes of PRRSV exist. However, prior to the present invention, the role of antigenic and genetic variation in the pathogenesis of PRRSV was not entirely clear.
The occurrence of PRRS in the U.S. has adversely affected the pig farming industry. Almost half of swine herds in swine-producing states in the U.S. are seropositive for PRRSV (Animal Pharm., 264:11 (Nov. 11, 1992)). In Canada, PRRS has been characterized by anorexia and pyrexia in sows lasting up to 2 weeks, late-term abortions, increased stillbirth rates, weak-born pigs and neonatal deaths preceded by rapid abdominal breathing and diarrhea. Work on the isolation of the virus causing PRRS, on a method of diagnosing PRRS infection, and on the development of a vaccine-against the PRRS virus has been published (see Canadian Patent Publication No. 2,076,744; PCT International Patent Publication No. WO 93/03760; PCT International Patent Publication No. WO 93/06211; and PCT International Patent Publication No. WO 93/07898).
There is also variability in the virulence of PRRSV in herds. Recently, a more virulent form of PRRS has been occurring with increased incidence in 3-8 week old pigs in the midwestern United States. Typically, healthy 3-5 week old pigs are weaned and become sick 5-7 days later. Routine virus identification methods on tissues from affected pigs have shown that swine influenza virus (SIV), pseudorabies virus (PRV), and Mycoplasma hyopneumoniae are not associated with this new form of PRRS. Originally termed proliferative interstitial pneumonia (PIP; see U.S. patent application Ser. No. 07/969,071), this disease has been very recently linked with PRRS, and the virus has been informally named the “Iowa strain” of PRRSV (see U.S. patent application Ser. No. 08/131,625).
Pessimism and skepticism has been expressed in the art concerning the development of effective vaccines against these porcine viruses (The Veterinary Record, Oct. 26, 1991). A belief that human influenza vaccine may afford some protection against the effects of PRRS and PNP exists (The Veterinary Record, Jul. 6, 1991).
Viral envelope proteins are known to be highly variable in many coronaviruses, such as feline infectious peritonitis virus and mouse hepatitis virus (Dalziel et al: Site-specific alteration of murine hepatitis virus type 4 peplomer glycoprotein E2 results in reduced neurovirulence. J. Virol., 59:464-471 (1986); Fleming et al: Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies. J. Virol., 58:869-875 (1986); Fiscus et al: Antigenic comparison of the feline coronavirus isolates; Evidence for markedly different peplomer glycoproteins. J. Virol., 61:2607-2613 (1987); Parker et al: Sequence analysis reveals extensive polymorphism and evidence of deletions within the E2 glycoprotein gene of several strains of murine hepatitis virus. Virology, 173:664-673 (1989)).
For example, a deletion or a mutation in the major envelope protein in coronaviruses can alter tissue tropism and in vivo pathogenicity. A mutation in a monoclonal antibody-resistant mutant of MHV has resulted in loss of its neurovirulence for mice (Fleming et al, 1986 supra). Porcine respiratory coronavirus (PRCV) is believed to be a deletion mutant of transmissible gastroenteritis virus (TGEV) in swine. The deletion in the PRCV genome may be in the 5′-end of the spike (S) gene of TGEV (Halbur et al, An overview of porcine viral respiratory disease. Proc. Central Veterinary Conference, pp. 50-59 (1993); Laude et al, Porcine respiratory coronavirus: Molecular features and virus-host interactions. Vet. Res., 24:125-150 (1993); Vaughn et al, Isolation and characterization of three porcine respiratory coronavirus isolates with varying sizes of deletions. J. Clin. Micro., 32:1809-1812 (1994)).
PRCV has a selective tropism for the respiratory tract and does not replicate in the gastrointestinal tract (Rasschaert et al, Porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions. J. Gen. Virol., 71:2599-2607 (1990); Laude et al, 1993 supra). In contrast, TGEV has a tropism for both respiratory and gastrointestinal tracts (Laude et al, 1993 supra).
Variation in antigenic and genetic relatedness among LDV isolates of varying pathogenicity is also known (Kuo et al, Lactate-dehydrogenase-elevating virus (LDV): subgenomic mRNAs, mRNA leader and comparison of 3′-terminal sequences of two LDV isolates. Virus Res., 23:55-72 (1992); Plagemann, LDV, EAV, and SHFV: A new group of positive stranded RNA viruses. Proc. Am. Assoc. Swine Practitioners, 4:8-15 (1992); Chen et al, Sequences of 3′ end of genome and of 5′ end of open reading frame 1a of lactate dehydrogenase-elevating virus and common junction motifs between 5′ leader and bodies of seven subgenomic mRNAs. J. Gen. Virol., 74:643-660 (1993)).
However, the present invention provides the first insight into the relationships between the open reading frames of the PRRSV genome and their corresponding effects on virulence and replication.
Further, a diagnosis of porcine reproductive and respiratory syndrome (PRRS) relies on compiling information from the clinical history of the herd, serology, pathology, and ultimately on isolation of the PRRS virus (PRRSV). Three excellent references reviewing diagnosis of PRRSV have been published in the last year (Van Alstine et al, “Diagnosis of porcine reproductive and respiratory syndrome,” Swine Health and Production, Vol. 1, No. 4 (1993), p. 24-28; Christianson et al, “Porcine reproductive and respiratory syndrome: A review.” Swine Health and Production, Vol. 1, No. 2 (1994), pp. 10-28 and Goyal, “Porcine reproductive and respiratory syndrome,” J. Vet. Diagn. Invest. 5:656-664 (1993)). PRRSV has also recently been shown to replicate in pulmonary alveolar macrophages by gold colloid immunohistochemistry (Magar et al (1993): Immunohistochemical detection of porcine reproductive and respiratory syndrome virus using colloidal gold. Can. J. Vet. Res., 57:300-304).
Clinical signs vary widely between farms, and thus, are not the most reliable evidence of a definitive diagnosis, except in the case of a severe acute outbreak in naive herds which experience abortion storms, increased numbers of stillborn pigs, and severe neonatal and nursery pig pneumonia. Presently, the most common clinical presentation is pneumonia and miscellaneous bacterial problems in 3-10 week old pigs. However, many PRRSV-positive herds have no apparent reproductive or respiratory problems.
Some herds evidence devastating reproductive failure, characterized by third-trimester abortions, stillborn pigs and weak-born pigs. Many of these herds also experience severe neonatal respiratory disease. Respiratory disease induced by PRRSV in 4-10 week-old pigs is also common and can be quite severe (Halbur et al, Viral contributions to the porcine respiratory disease complex. Proc. Am. Assoc. Swine Pract. (1993), pp. 343-350). Clinical PRRSV outbreaks are frequently followed by bacterial pneumonia, septicemia, or enteritis. Thus, it has been difficult to obtain an acceptably rapid and reliable diagnosis of infection by PRRSV, prior to the present invention.
The pig farming industry has been and will continue to be adversely affected by these porcine reproductive and respiratory diseases and new variants thereof, as they appear. PRRSV is a pathogen of swine that causes economic losses from reproductive and respiratory diseases. Economic losses from PRRS occur from loss of pigs from abortions, stillborn pigs, repeat breeding, pre-weaning and postweaning mortality, reduced feed conversion efficiency, increased drug and labor cost and have been estimated to cost approximately $236 per sow in addition to loss of profits (Polson et al., Financial implications of mystery swine disease (MSD), Proc. Mystery Swine Disease Committee Meeting, Denver, Colo., 1990, pp. 8-28). This represents a loss of $23,600 for a 100 sow herd or $236,000 for a 1000 sow herd.
PRRSV causes additional losses from pneumonia in nursery pigs. However, the exact economic losses from PRRSV-associated pneumonia are not known. PRRSV is an important cause of pneumonia in nursery and weaned pigs. Reproductive disease was the predominant clinical outcome of PRRSV infections during the past few years. Respiratory disease has now become the main problem associated with PRRSV.
Surprisingly, the market for animal vaccines in the U.S. and worldwide is larger than the market for human vaccines. Thus, there exists an economic incentive to develop new veterinary vaccines, in addition to the substantial public health benefit which is derived from protecting farm animals from disease.